The small GTPases of the Ras-related Rho family have been implicated in the progression of human tumors to an invasive metastatic state. Among the key regulators of these proteins are the GTPase activating proteins (GAPs), which promote the inactivation of Rho GTPases. However, relatively little is known regarding the regulation of the GAPs. Dr. Settleman has been investigating the biological function of one of the major RhoGAPs, known as p190 RhoGAP, for more than 10 years. Recently, collaborative efforts between Drs. Settleman and Ligeti have revealed that phospholipids regulate the GTPase substrate specificity of p190 RhoGAP--inhibiting its activity toward the Rho GTPase, while promoting its activity toward the Rac GTPase. This finding indicates a novel regulatory mechanism for the Rho GTPases--altering the specificity of GAPs for their GTPase substrates. The newly proposed studies will examine the biochemical nature of the phospholipid effects on p190 RhoGAP, and include an analysis of the major classes of cellular phospholipids that could potentially regulate p190 RhoGAP, a mutational analysis to identify the lipid-interacting domain, and studies to address the mechanism by which lipids affect GAPGTPase interactions. In addition, the role of PKC-mediated phosphorylation of the catalytic GAP domain of p190 in regulating GTPase substrate specificity will be examined. It is anticipated that these collective studies will reveal important insights into this novel regulatory mechanism for Rho GTPases that could potentially extend to some of the many other classes of related GTPases that play a role in tumor initiation and progression.